Project Summary The cell type that undergoes cancer initiation, or ?cell of origin,? plays a key role in determining tumor properties such as molecular and histopathological subtype as well as treatment response and disease outcome. In the case of prostate cancer, the cell of origin may be highly relevant for whether it is indolent or aggressive, and consequently the analysis of cell types of origin may lead to identification of biomarkers that can guide therapy. Over the past ten years, our laboratory has made considerable progress in understanding prostate cell types of origin through studies of the progenitor populations, lineage relationships, and transcriptional regulators of the prostate epithelium during normal development as well as cancer initiation and progression. In preliminary studies for this proposal, we have performed single-cell RNA sequencing to examine the heterogeneity of prostate epithelial cells in the benign mouse and human prostate. Notably, these analyses have revealed several different luminal populations that have not been previously identified, and may represent distinct cell types of origin. Furthermore, we have used computational systems approaches to identify candidate master regulator (driver) genes for these novel luminal populations. Finally, we have established three-dimensional organoid culture conditions to investigate whether specific mouse and human epithelial cell populations are cell types of origin for prostate cancer. Based on our preliminary data, we hypothesize that analysis of the origin and drivers of specific epithelial populations in the prostate will elucidate cell types of origin for prostate cancer as well as their roles in specifying tumor phenotypes. To investigate this hypothesis, we will pursue an innovative combination of in vivo, ex vivo, molecular, and computational systems approaches using genetically-engineered mouse models and human prostate tissue. We propose three specific aims: (1) Investigation of epithelial heterogeneity in the developing and adult prostate using single-cell transcriptomics to examine the origin and lineage relationships of distinct epithelial populations and identify drivers that specify these populations; (2) Analysis of cell of origin in mouse and human prostate using organoid culture approaches to determine whether distinct luminal and basal epithelial populations can give rise to tumors after oncogenic transformation and identify candidate master regulators of this process; and (3) Investigation of epithelial heterogeneity in mouse and human prostate cancer using cross-species single-cell analyses to identify conserved drivers of tumor heterogeneity. Taken together, our studies will provide fundamental molecular and functional insights into prostate epithelial heterogeneity during development and tumorigenesis, and will have important implications for understanding the origins of human prostate cancer and its treatment.